Summary Nearly 2 million people live with a lost limb in the United States alone. To date, there is no way to promote the ability to regenerate limbs in humans. Yet the distal limb region, the digit tip, can undergo perfect regeneration. Mammalian digit tip regeneration is a remarkable example of epimorphic regeneration. Upon amputation of the digit tip in mice and humans, undifferentiated mesenchyme, called the blastema, forms underneath the wound epidermis, through a process that histologically mirrors amphibian limb regeneration. This blastema ultimately resolves into a perfectly regenerated tip, including nail and bone. Although this regeneration ability is limited to the distal tip of the digits, understanding this phenomenon can provide unique insights about regeneration of more proximal amputations. Currently, little is known about the origin and characteristics of the mammalian blastema, the key structure for the successful digit tip regeneration. To what extent the cells at the amputation stump undergo de- differentiation/trans-differentiation during the blastema formation and subsequent regeneration remains elusive in lower vertebrates and indeed, unknown in mammals. Our preliminary results in mice show that fibroblasts can migrate into the amputation site to form the blastema, and then alter their fate into the bone lineage to assist in tip regeneration. This raises an exciting possibility that fibroblasts, abundant cells within human body, may have the potential to create a regeneration-competent blastema. We hypothesize that exogenous fibroblasts are competent to give rise to blastema that ultimately differentiates into new digit bone and surrounding mesenchyme. A major challenge in addressing this hypothesis and other essential questions about tip regeneration, is the well-known caveat in the field that the mouse digit tip is extremely small. We propose a step-wise project: 1) Explore the feasibility of using rats as a preclinical model for digit tip regeneration; 2) transplant rat (and possibly human) fibroblasts into the rat digit tip (30 times larger than mouse tip). During the R61 phase, we will comprehensively characterize the rat digit tip regeneration using histochemistry and scRNA seq, and begin testing the ability of exogenous fibroblasts to undergo osteolineage conversion upon transplantation into the digit tip. We will trace the behavior of fibroblasts to determine their contribution to the digit tip regeneration upon distal and proximal amputations in rats. In R33 phase, we will isolate blastemal cells from rat digits and examine epigenetic changes seen during the blastemal formation in rats using ATAC seq. In parallel, we will test how exogenous human fibroblasts promotes digit tip regeneration using immunodeficient rats as recipients. These experiments will help establish an effective cellular therapy to create, or at the least, promote blastema formation in mammals ultimately for limb regeneration.